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D K Ghosh

Publications and source records attributed to D K Ghosh.

At least 19 recordsLinked to original sources
PubMed2005

Multi-frequency EPR and Mössbauer spectroscopic studies on freeze-quenched reaction intermediates of nitric oxide synthase.

It is believed by analogy to chloroperoxidase (CPO) from Caldariomyces fumago that the electronic structure of the intermediate iron-oxo species in the catalytic cycle of nitric oxide synthase (NOS) corresponds to an iron(IV) porphyrin-pi -cation radical. Such species can also be produced by the reaction of ferric NOS with external oxidants within the shunt pathway. We present multi-frequency EPR (9.6, 94, 285 GHz) and Mössbauer spectroscopic studies on freeze-quenched intermediates of the oxygenase domain of nitric oxide synthase which has reacted with peroxy acetic acid within 8-200 ms. The intermediates of the oxygenase domain of both the cytokine inducible NOS (iNOSox) and the neuronal NOS (nNOSox) show an organic radical signal in the 9.6-GHz spectrum overlapping with the spectrum of an unknown species with g-values of 2.24, 2.23 and 1.96. Using 94- and 285-GHz EPR the organic radical signal is assigned to a tyrosine radical on the basis of g-values (i.e. Tyr*562 in nNOSox and Tyr*341 in iNOSox). Mössbauer spectroscopy of (57)Fe-labeled unreacted nNOSox shows a ferric low-spin heme-iron (delta = 0.38 mms(-1), deltaE(Q) = 2.58 mms(-1)). The reaction of nNOSox with peroxy acetic acid for 8 ms leads to the disappearance of the magnetic background characteristic for native nNOSox and a new species with delta = 0.27 mms(-1) and deltaE(Q) = 2.41 mms(-1) is detected at 4.2 K which does not resemble the parameters typical for a Fe(IV) center. It is proposed that this intermediate species corresponds to a ferric low-spin species which magnetically couples to an amino acid radical (presumably Trp*409).

Binding Sites↗
PubMed2004

Separation of thoraco-omphalopagus Siamese twin.

A pair of female thoraco-omphalopagus twins, with two pairs of lungs, common diaphragm and separate pleural cavities was separated at the age of 5(1/2) months after a parasitic relationship had developed between them. Before separation both the babies developed recurrent respiratory tract infection and frequent diarrhea. They were treated with medical therapy and made fit for anesthesia. The combined weight of the twins was 4(1/2) kg at birth and the combined weight at the time of separation was 9(1/2) kg. One baby was smaller in growth compared to the other baby. The closure of anterior abdominal wall was difficult in both babies. However, we could close it without use of any biodegradable patches or tissue expander. Both the babies survived and are doing well after 3 months of separation.

Female↗
PubMed2004

High pressure fourier transform infrared (FT-IR) spectroscopic studies on inducible nitric oxide (NO) synthase active site: a comparison to cytochrome p450CAM.

High pressure Fourier transform infrared (FT-IR) spectroscopy is performed for the first time to analyse the active site of inducible nitric oxide synthase (iNOSox) using the carbon monoxide (CO) heme iron ligand stretch mode (nuCO) as spectroscopic probe. A membrane-driven sapphire anvil high-pressure cell is used. Three major conformational substates exist in substrate-free iNOSox which are characterized by nuCO at approximately 1936, 1945 and 1952 cm(-1). High pressure favors the 1936 cm(-1) substate with a volume difference to the 1945 substate of approximately -21 cm3/mol. The pressure induced cytochrome P420 formation with a reaction volume of approximately -80 cm3/mol is observed. Arginine binding produces a very low nuCO at approximately 1905 cm(-1) caused by the H-bond from the substrate to CO. nuCO for the substates in the substrate-free and arginine-bound proteins shift linearly with pressure which is qualitatively similar to the observation on cytochrome P450cam. The slightly smaller positive slope of the shift in substrate-free iNOSox compared to substrate-free P450cam is interpreted as a slightly lesser compressible heme pocket. In contrast, the significant slower negative slope for arginine-bound iNOSox compared to camphor-bound P450cam results from the different kind of interactions to the CO ligand (electrostatic interaction in P450cam, H-bond in iNOSox).

Animals↗
PubMed2002

Beryllium: an easily scavenged metal ion in the environment.

This study was specifically aimed to determine the levels of beryllium in environmental samples near the vicinity of the beryllium metal plant (BMP). Air particulate samples collected at the BMP site, in the non-monsoon and monsoon seasons, showed an average beryllium concentration of 0.3 and 0.1 ng m(-3) respectively, where as rain water samples showed the beryllium values in the range of 0.01-0.2 ng ml(-1). The suspended particulate matter (dust load) at the site studied was 570 and 250 microg m(-3) in the non-monsoon and monsoon seasons respectively. The results obtained show that, in the environment, 80% of the total beryllium present is removed by rain.

Beryllium↗
PubMed2002

Immobilisation of beryllium in solid waste (red-mud) by fixation and vitrification.

The objective of this study was to obtain information on the immobilization of beryllium (Be) in solid waste generated in the extraction process of beryllium from its ore, Beryl. This solid waste, termed red-mud, contains oxides of iron, aluminium, calcium, magnesium and beryllium. The red-mud waste contains beryllium at levels above the permissible limit, which prevents its disposal as solid waste. The total beryllium content in the red-mud analysed showed value ranging from 0.39 to 0.59% Be The studies showed that 50% of the total beryllium in red-mud can be extracted by water by repeated leaching over a period of 45 days. The cement mix, casting into cement blocks, was subjected to leachability studies over a period of 105 days and immobilization factor (IF factor) was determined. These IF values, of the order of 102, were compared with those obtained by performing leachability study on vitrified red-mud masses produced at different temperature conditions. Direct heating of the red-mud gave the gray coloured, non-transparent vitreous mass (as 'bad glass') showed effective immobilisation factor for beryllium in red-mud of the order of 10(4).

Beryllium↗
PubMed2001

Characterization of key residues in the subdomain encoded by exons 8 and 9 of human inducible nitric oxide synthase: a critical role for Asp-280 in substrate binding and subunit interactions.

Human inducible nitric oxide synthase (iNOS) is active as a dimer of two identical subunits. Each subunit has an amino-terminal oxygenase domain that binds the substrate l-Arg and the cofactors heme and tetrahydrobiopterin and a carboxyl-terminal reductase domain that binds FMN, FAD, and NADPH. We previously demonstrated that a subdomain in the oxygenase domain encoded by exons 8 and 9 is important for dimer formation and NO synthesis. Further, we identified Trp-260, Asn-261, Tyr-267, and Asp-280 as key residues in that subdomain. In this study, using an Escherichia coli expression system, we produced, purified, and characterized wild-type iNOS and iNOS-Ala mutants. Using H(2)O(2)-supported oxidation of N(omega)-hydroxy-l-Arg, we demonstrate that the iNOS mutants' inabilities to synthesize NO are due to selective defects in the oxygenase domain activity. Detailed characterization of the Asp-280-Ala mutant revealed that it retains a functional reductase domain, as measured by its ability to reduce cytochrome c. Gel permeation chromatography confirmed that the Asp-280-Ala mutant exists as a dimer, but, in contrast to wild-type iNOS, urea-generated monomers of the mutant fail to reassociate into dimers when incubated with l-Arg and tetrahydrobiopterin, suggesting inadequate subunit interaction. Spectral analysis reveals that the Asp-280-Ala mutant does not bind l-Arg. This indicates that, in addition to dimerization, proper subunit interaction is required for substrate binding. These data, by defining a critical role for Asp-280 in substrate binding and subunit interactions, give insights into the mechanisms of regulation of iNOS activity.

Aspartic Acid↗
PubMed2001

Beryllium concentrations in ambient air and its source identification. A case study.

Beryllium concentrations in atmospheric particulate and soil samples in and around a Beryllium Processing Facility (BPF) have been measured. The mean air concentration level of beryllium in and around the fence line of the BPF is 0.48 +/- 0.43 ng m(-3) (n = 397) and is mostly influenced by diurnal and seasonal changes. The observed air concentration levels were well below the prescribed ambient air quality (AAQ) standard of 10 ng m(-3). The soil concentration levels of beryllium in the study area were found to be in the range of 1.42-2.75 microg g(-1). The mass median aerodynamic diameter (MMAD) of beryllium aerosols in ambient air was found to be 6.9 microm. Source identification using the Enrichment Factor (EF) approach indicates soil as the predominant contributory source for air concentrations at the site.

Aerosols↗
PubMed2001

Host response to infection: the role of CpG DNA in induction of cyclooxygenase 2 and nitric oxide synthase 2 in murine macrophages.

Depending on sequence, bacterial and synthetic DNAs can activate the host immune system and influence the host response to infection. The purpose of this study was to determine the abilities of various phosphorothioate oligonucleotides with cytosine-guanosine-containing motifs (CpG DNA) to activate macrophages to produce nitric oxide (NO) and prostaglandin E(2) (PGE(2)) and to induce expression of NO synthase 2 (NOS2) and cyclooxygenase 2 (COX2). As little as 0.3 microg of CpG DNA/ml increased NO and PGE(2) production in a dose- and time-dependent fashion in cells of the mouse macrophage cell line J774. NO and PGE(2) production was noted by 4 to 8 h after initiation of cultures with the CpG DNA, with the kinetics of NO production induced by CpG DNA being comparable to that induced by a combination of lipopolysaccharide and gamma interferon. CpG DNA-treated J774 cells showed enhanced expression of NOS2 and COX2 proteins as determined by immunoblotting, with the relative potencies of the CpG DNAs generally corresponding to those noted for the induction of NO and PGE(2) production as well as to those noted for the induction of interleukin-6 (IL-6), IL-12, and tumor necrosis factor. Extracts from CpG DNA-treated cells converted L-arginine to L-citrulline, but the NOS inhibitor N(G)-monomethyl-L-arginine (NMMA) inhibited this reaction. The COX2-specific inhibitor NS398 inhibited CpG DNA-induced PGE(2) production and inhibited NO production to various degrees. The NOS inhibitors NMMA, 1400W, and N-iminoethyl-L-lysine effectively blocked NO production and increased the production of PGE(2) in a dose-dependent fashion. Thus, analogues of microbial DNA (i.e., CpG DNA) activate mouse macrophage lineage cells for the expression of NOS2 and COX2, with the production of NO and that of PGE(2) occurring in an interdependent manner.

Adjuvants, Immunologic↗
PubMed2000

FT-Infrared spectroscopic studies of the iron ligand CO stretch mode of iNOS oxygenase domain: effect of arginine and tetrahydrobiopterin.

The iron ligand CO stretch vibration mode of the inducible nitric oxide synthase oxygenase domain (iNOSox) has been studied from 20 to 298 K. iNOSox in the absence of arginine reveals a temperature-dependent equilibrium of two major conformational substates with CO stretch bands centered at about 1945 and 1954 cm(-)(1). This behavior is not qualitatively changed when tetrahydrobiopterin (H(4)B) is bound. Arginine binding changes significantly the spectrum by formation of a sharp CO stretch mode band at about 1905 cm(-)(1) and indicates the formation of a hydrogen bond to the CO ligand. For temperatures lower than 250 K, the stretch vibration frequency decreases almost linearly with decreasing temperature and indicates that the coupling between the CO ligand and the arginine/protein in the active site via the hydrogen bond is very strong. Flashphotolysis of the CO ligand carried out at 25 K revealed the CO stretch mode of the photodissociated CO ligand trapped in the heme pocket. There is a negative linear relation between the stretch vibration frequencies of the photodissociated and the iron-bound CO indicating that the photodissociated ligand stays near the heme.

Animals↗
PubMed1999

Inducible nitric oxide synthase: role of the N-terminal beta-hairpin hook and pterin-binding segment in dimerization and tetrahydrobiopterin interaction.

The oxygenase domain of the inducible nitric oxide synthase (iNOSox; residues 1-498) is a dimer that binds heme, L-arginine and tetrahydrobiopterin (H(4)B) and is the site for nitric oxide synthesis. We examined an N-terminal segment that contains a beta-hairpin hook, a zinc ligation center and part of the H(4)B-binding site for its role in dimerization, catalysis, and H(4)B and substrate interactions. Deletion mutagenesis identified the minimum catalytic core and indicated that an intact N-terminal beta-hairpin hook is essential. Alanine screening mutagenesis of conserved residues in the hook revealed five positions (K82, N83, D92, T93 and H95) where native properties were perturbed. Mutants fell into two classes: (i) incorrigible mutants that disrupt side-chain hydrogen bonds and packing interactions with the iNOSox C-terminus (N83, D92 and H95) and cause permanent defects in homodimer formation, H(4)B binding and activity; and (ii) reformable mutants that destabilize interactions of the residue main chain (K82 and T93) with the C-terminus and cause similar defects that were reversible with high concentrations of H(4)B. Heterodimers comprised of a hook-defective iNOSox mutant subunit and a full-length iNOS subunit were active in almost all cases. This suggests a mechanism whereby N-terminal hooks exchange between subunits in solution to stabilize the dimer.

Amino Acid Sequence↗
PubMed1999

N-terminal domain swapping and metal ion binding in nitric oxide synthase dimerization.

Nitric oxide synthase oxygenase domains (NOS(ox)) must bind tetrahydrobiopterin and dimerize to be active. New crystallographic structures of inducible NOS(ox) reveal that conformational changes in a switch region (residues 103-111) preceding a pterin-binding segment exchange N-terminal beta-hairpin hooks between subunits of the dimer. N-terminal hooks interact primarily with their own subunits in the 'unswapped' structure, and two switch region cysteines (104 and 109) from each subunit ligate a single zinc ion at the dimer interface. N-terminal hooks rearrange from intra- to intersubunit interactions in the 'swapped structure', and Cys109 forms a self-symmetric disulfide bond across the dimer interface. Subunit association and activity are adversely affected by mutations in the N-terminal hook that disrupt interactions across the dimer interface only in the swapped structure. Residue conservation and electrostatic potential at the NOS(ox) molecular surface suggest likely interfaces outside the switch region for electron transfer from the NOS reductase domain. The correlation between three-dimensional domain swapping of the N-terminal hook and metal ion release with disulfide formation may impact inducible nitric oxide synthase (i)NOS stability and regulation in vivo.

Amino Acid Sequence↗
PubMed1998

Structure of nitric oxide synthase oxygenase dimer with pterin and substrate.

Crystal structures of the murine cytokine-inducible nitric oxide synthase oxygenase dimer with active-center water molecules, the substrate L-arginine (L-Arg), or product analog thiocitrulline reveal how dimerization, cofactor tetrahydrobiopterin, and L-Arg binding complete the catalytic center for synthesis of the essential biological signal and cytotoxin nitric oxide. Pterin binding refolds the central interface region, recruits new structural elements, creates a 30 angstrom deep active-center channel, and causes a 35 degrees helical tilt to expose a heme edge and the adjacent residue tryptophan-366 for likely reductase domain interactions and caveolin inhibition. Heme propionate interactions with pterin and L-Arg suggest that pterin has electronic influences on heme-bound oxygen. L-Arginine binds to glutamic acid-371 and stacks with heme in an otherwise hydrophobic pocket to aid activation of heme-bound oxygen by direct proton donation and thereby differentiate the two chemical steps of nitric oxide synthesis.

Animals↗
PubMed1998

Stopped-flow analysis of CO and NO binding to inducible nitric oxide synthase.

The oxygenase domain (amino acids 1-498) of inducible nitric oxide synthase (iNOSox) is a hemeprotein that binds L-arginine (L-Arg) and tetrahydrobiopterin (H4B). During NO synthesis, the heme iron must bind and activate O2, but it also binds self-generated No to form an inactive complex. To better understand how L-Arg and H4B affect heme iron function in iNOSox, we utilized stopped-flow spectroscopy to study heme reactivity with CO and NO and the properties of the resulting CO and NO complexes. CO and NO binding to ferrous and ferric (NO only) iNOSox and subsequent complex stability was studied under four conditions: in the absence of L-Arg and H4B and in the presence of either or both molecules. Ferric iNOSox without L-Arg or H4B was dimeric and contained low-spin heme iron, while in H4B- or L-Arg-saturated iNOSox, the heme iron was partially or almost completely high-spin, respectively. In the presence of L-Arg or H4B, the rate of CO binding to ferrous iNOSox was slowed considerably, indicating that these molecules restrict CO access to the heme iron. In contrast, rates of NO binding were minimally affected. Under all conditions, the off rates for CO and NO were very high as compared to other hemeproteins. The six-coordinate FeII-CO and -NO complexes that initially formed were unstable and converted either slowly (CO) or quickly (NO) to their respective 5-coordinate complexes. However, this transition was largely prevented by either L-Arg or H4B and was reversed upon air oxidation of the complex in the presence of these molecules. Thus, H4B and L-Arg both promote a conformational change in the distal heme pocket of iNOSox that can greatly reduce ligand access to the heme iron. The ability of H4B and L-Arg to prevent formation of a five-coordinate heme Fe-NO complex, along with the high off rates observed for NO, help explain why iNOS can remain active despite forming a complex with NO during its normal catalysis.

Binding Sites↗
PubMed1997

The structure of nitric oxide synthase oxygenase domain and inhibitor complexes.

The nitric oxide synthase oxygenase domain (NOSox) oxidizes arginine to synthesize the cellular signal and defensive cytotoxin nitric oxide (NO). Crystal structures determined for cytokine-inducible NOSox reveal an unusual fold and heme environment for stabilization of activated oxygen intermediates key for catalysis. A winged beta sheet engenders a curved alpha-beta domain resembling a baseball catcher's mitt with heme clasped in the palm. The location of exposed hydrophobic residues and the results of mutational analysis place the dimer interface adjacent to the heme-binding pocket. Juxtaposed hydrophobic O2- and polar L-arginine-binding sites occupied by imidazole and aminoguanidine, respectively, provide a template for designing dual-function inhibitors and imply substrate-assisted catalysis.

Amino Acid Sequence↗
PubMed1997

Characterization of the inducible nitric oxide synthase oxygenase domain identifies a 49 amino acid segment required for subunit dimerization and tetrahydrobiopterin interaction.

The oxygenase domain of inducible NO synthase (residues 1-498, iNOSox) is the enzyme's catalytic center. Its active form is a homodimer that contains heme and tetrahydrobiopterin (H4biopterin) and binds l-arginine [Ghosh, D. K., & Stuehr, D. J. (1995) Biochemistry 34, 801]. To help identify protein residues involved in prosthetic group and dimeric interaction, we expressed H4biopterin-free iNOSox in Escherichia coli. The iNOSox was 80% dimeric but contained a low-spin heme iron that bound DTT as a sixth ligand. The iNOSox bound H4biopterin or L-arginine with high affinity, which displaced DTT from the heme and caused spectral changes consistent with a closing up of the heme pocket. The H4biopterin-replete iNOSox could catalyze conversion of Nomega-hydroxyarginine to citrulline and NO in a H2O2-supported reaction. Limited trypsinolysis of the H4biopterin-free iNOSox dimer cut the protein at a single site in its N-terminal region (K117). H4biopterin protected against the cleavage whereas l-arginine did not. The resulting 40 kDa protein contained thiol-ligated low-spin heme, was monomeric, catalytically inactive, showed no capacity to bind H4biopterin or l-arginine, and did not dimerize when provided with these molecules, indicating that residues 1-117 were important for iNOSox dimerization and H4biopterin/l-arginine interaction. A deletion mutant missing residues 1-114 was partially dimeric but otherwise identical to the 40 kDa protein regarding its spectral and catalytic properties and inability to respond to l-arginine and H4biopterin, whereas a deletion mutant missing residues 1-65 was equivalent to wild-type iNOSox, narrowing the region of importance to amino acids 66-114. Mutation of a conserved cysteine in this region (C109A) decreased H4biopterin affinity without compromising iNOSox dimeric structure, L-arginine binding, or catalytic function. These results suggest that residues 66-114 of iNOSox are involved in productive H4biopterin interaction and subunit dimerization. H4biopterin binding appears to stabilize the protein structure in this region, and through doing so activates iNOS for NO synthesis.

Amino Acid Sequence↗
PubMed1997

Mutagenesis of acidic residues in the oxygenase domain of inducible nitric-oxide synthase identifies a glutamate involved in arginine binding.

The oxygenase domain of the mouse cytokine-inducible nitric-oxide synthase (iNOSox, amino acids 1-498) binds heme, tetrahydrobiopterin, and the substrate Arg and is the domain responsible for catalyzing nitric oxide synthesis and maintaining the enzyme's active dimeric structure. To further understand iNOSox structure-function, we carried out alanine point mutagenesis on 15 conserved acidic residues located within a region of iNOSox (amino acids 352-473) that shares sequence homology with the pterin-binding module in dihydrofolate reductases and may be important for iNOSox subunit dimerization and/or Arg binding. Five point mutants were identical or nearly identical to wild-type, while 10 exhibited a range of defects that included low heme content (2), heme ligand instability (2), defective dimerization (2), and poor Arg and/or tetrahydrobiopterin binding (4). Mutations that caused defective tetrahydrobiopterin binding were also associated with other defects. In contrast, two mutants (E371A and D376A) exhibited an exclusive defect in Arg binding. These mutants were dimeric, indicating that dimerization of iNOSox in Escherichia coli does not require Arg. In one case (E371A), the defect in Arg binding was absolute, as assessed by spectral perturbation, radioligand binding, and catalytic studies. We conclude that mutagenesis of conserved acidic residues within this region of iNOSox can lead to exclusive defects in dimerization and in Arg binding. Modeling considerations predict that the E371 carboxylate may participate in Arg binding by interacting with its guanidine moiety.

Alanine↗
PubMed1997

Mercury and organomercurial resistance in bacteria isolated from freshwater fish of wetland fisheries around Calcutta.

Mercury-resistant bacteria belonging to the genera Bacillus, Escherichia, Klebsiella, Micrococcus, Pseudomonas, Salmonella, Sarcina, Shigella, Staphylococcus and Streptococcus were isolated from gills and guts of fresh water fish collected from wetland fisheries around Calcutta, India, contaminated with mercury compounds. The total number of bacteria, as well as Hg-resistant bacteria, were always higher in guts than gills. Bottom-dwelling fish contained higher number of bacteria, including Hg-resistant bacteria, than surface and middle water dwelling fish. They belonged either to narrow-spectrum or to broad-spectrum Hg-resistant groups and they also possessed other heavy metal and antibiotic resistant properties. In the presence of toxic levels of HgCl(2), phenylmercuric acetate (PMA) and methylmercuric chloride (MMC), the lag in growth of the bacterial strains gradually increased with increasing concentration of Hg-compounds. Narrow-spectrum Hg-resistant bacterial strains volatilized only HgCl(2) from the liquid medium in the range of 64-89%, whereas the broad-spectrum group exhibited a high level of HgCl(2) (80-94%), PMA (72-84%) and MMC (64-80%) volatilizing capacity with inducible mercuric reductase and organomercurial lyase enzyme activities in their cell-free extracts. Cell-free extracts prepared from narrow-spectrum Hg-resistant bacterial strains induced by HgCl(2) exhibited Hg(+2)-dependent NADPH oxidation, indicating the presence of only mercuric reductase enzyme.

Journal Article↗